The estimation of metallothionein (MT) levels in a biological sample is required to study the physiological role or the induction of MT, which is a multifunctional protein. MT contains 61 amino acids with 20 homologous cysteines. The high selectivity, sensitivity and stability of N-[4-(dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM) for the determination of SH groups was very useful for the determination of MT. The detection limit of MT by this method was 10 ng/0.5 ml. This method made it possible to measure MT levels in normal tissues in combination with gel filtration chromatography. The age-dependent change in MT levels in normal rat tissues was also investigated by the present method. MT levels in normal rat tissues vary with age. It is though that this fact is correlated with the biological roles of MT.
NCC-CO-411, an anti-carcinoembryonic antigen (CEA) monoclonal antibody (MoAb) showed a dose-dependent reactivity with standard CEA, but did not bind to the CEA anchoring with cell membranes. When LS 174T cells, one of the CEA-producing cells, were treated with phosphatidylinositol-specific phospholipase C, NCC-CO-411 MoAb recognized the released CEA containing glycosylphosphatidylinositol as well as the standard CEA. The binding activity of NCC-CO-411 MoAb with CEA was also demonstrated by the immunostaining of the LS 174T colorectal tumor xenograft tissues. The biodistribution study showed that NCC-CO-411 MoAb was mainly taken up by tumor and kidney, while the anti-hCEA MoAb was largely concentrated in liver and spleen rather than in the tumor. These results suggest that NCC-CO-411 MoAb recognizes the released CEA and gives an excellent tumor image in LS 174T tumor-bearing mice.
Plasmids carrying the groE promoter with a superhelical density of 0 to -0.091 were used for in vitro transcription with RNA polymerase holoenzyme containing σ32. The promoter activity of the groE gene in vitro on the plasmid DNA was decreased to 100-fold when the superhelical density of the template DNA decreased from -0.059 to 0. As the superhelical density of DNA inside the cells was estimated to be approximately -0.025, it seems likely that the increase in the linking number of template DNA caused by stress which induces heat shock response may not stimulate the groE promoter activity in vivo on the chromosomal DNA. At higher concentrations of salt, the dependence of the groE promoter activity on negative supercoiling was even more apparent. Since melting of the DNA duplex is inhibited by high salt concentrations, DNA supercoiling may facilitate melting of the groE promoter by an RNA polymerase holoenzyme containing σ32.
The effects on pilocarpine-induced saliva secretion by a hot aqueous extract of Byakko-ka-ninjin-to (BN), its constituents, rhizomes of Anemarrhena asphodeloides, three saponins (pseudoproto-timosaponin-AIII (An-S-1), proto-timosaponin-AIII (An-S-2) and timosaponin-AIII (An-S-3)) and calcium were examined in streptozocin (STZ)-induced diabetic and normal mice. The hot aqueous extracts of BN (250 and 500 mg/kg, i.p) and Anemarrhena (170 and 340 mg/kg, i.p.) significantly promoted salivary flow in the diabetic animals, but suppressed it in the normal controls. An-S-2 and An-S-3 but not An-S-1 (10 mg/kg, i.p.), significantly promoted salivary flow in the diabetic animals. The potency order was An-S-3»An-S-2»extract. The hot aqueous extracts of BN and Anemarrhena increased the protein content of saliva in a dose-dependent manner. Combination of An-S-3 (0.1 mg/kg, i.p.) with CaCl2 (2 and 4 mg/kg, i.p.) potentiated salivary flow compared with the respective effect of each on its own. These results demonstrated that 1) An-S-3 was mainly responsible for saliva secretion of the hot aqueous extract, and 2) the effect of An-S-3 was potentiated by combination with calcium, suggesting combined effects of Byakko-ka-ninjin-to containing Anemarrhena asphodeloides and gypsum fiber (calcium).
The effects of the chronic administration of bopindolol on the binding characteristics of [3H]CGP12177 and [3H]prazosin to cardiac α1H-, α1L-, β1- and β2-adrenoceptor subtypes of spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were compared with those of two other β-blockers, atenolol and propranolol. Bopindolol (1 and 3 mg/kg/d), atenolol (50 mg/kg/d) and propranolol (60 mg/kg/d) were given to 10-week-old SHR for 12 weeks. The changes in Kd and Bmax values of the myocardium of SHR treated without and with those drugs were assessed by Scatchard analysis, and the ratio and Bmax values of the β1- and β2-adrenoceptor subtypes were also calculated from displacemental curves using ICI 118, 551. The systolic blood pressure in SHR was dose-dependently lowered by the administration of dopindolol, and was also lowered by the administration of atenolol and propranolol. The Bmax values of β1- and β2-adrenoceptors were lowered by the administration of bopindolol (1 and 3 mg/kg/d) without any changes in the Kd values or the ratio of β1- and β2-adrenoceptors. Propranolol lowered 3-fold the affinity to the β-adrenoceptor. On the other hand, the Kd and Bmax values of α1H- and α1L-adrenoceptor subtypes (high and low affinity binding sites for [3H]prazosin) were not changed by these drugs. These findings suggest that bopidolol had a beneficial effect on β-adrenoceptors in the membranes of cardiac muscles of SHR, simplying that these effects may contribute to lowering hypertension.
Shosaiko-to (Xiao-chai-hu-tang, SHO), a Kampo medicine, was prepared by decocting a prescription of 7 kinds of crude drugs, namely Bupleuri Radix, Pinelliae Tuber, Scutellariae Radix, Zizyphi Fructus, Ginseng Radix, Glycyrrhizae Radix and Zingiberis Rhizoma. Previously, we reported that the effect of the orally administered SHO in augmenting natural killer (NK) activity in the peripheral blood was attributed to the acidic polysaccharide fraction.To characterize the active components in the crude materials in SHO, the effects of extracts and various fractions were investigated by oral administration. The extract of Zizyphi Fructus, Zingiberis Rhizoma, Scutellariae Radix, Glycyrrhizae Radix and Pinelliae Tuber augmented NK activity by oral administration. The high weight molecular fraction of Zizyphi Fructus was the most effective in augmenting NK activity. Thus, we obtained an active polysaccharide fraction from the high weight molecular fraction of Zizyphi Fructus. This polysaccharide fraction with a high molecular weight of approximately 43000 contained 54.7% carbohydrate, 61.8% uronic acid and 20.9% protein. The sugar moiety was composed of rhamnose, arabinose, xylose, fucose, mannose, galactose, glucose and galacturonic acid in molar ratios of 28 : 59 : 11 : 9 : 7 : 32 : 20 : 100.
We examined the effects of KW-3902 (8-(noradamantan-3-yl)-1, 3-dipropylxanthine), an adenosine A1-receptor antagonist, on urine volume and urinary excretions of various electrolytes in saline-loaded rats, as compared with those of furosemide, trichlormethiazide (TCM), acetazolamide and amiloride. KW-3902 at doses of 0.001-1 mg/kg (p.o.) significantly increased urine volume and excretions of sodium, calcium, magnesium, chloride and bicarbonate. In addition, KW-3902 shifted urine pH to alkaline and decreased free water reabsorption. KW-3902 did not induce kaliuresis, whereas furosemide (30 mg/kg, p.o.), TCM (1 mg/lg, p.o.) and acetazolamide (25 mg/kg, p.o.) induced kaliuresis. In the KW-3902-treated group, the increases in bicarbonate excretion and urine pH were less prominent than those induced by acetazolamide, and the excretions of sodium, calcium, magnesium and chloride were similar to those induced by furosemide. The present results suggest that the adenosine A1-receptor antagonist exhibits diuresis by the inhibited reabsorption of electrolytes, not only at the proximal tubule but also at the distal tubule.
Effects on zinc (Zn) and copper (Cu) on cadmium (Cd) accumulation were investigated in LLC-PK1 cells. Coincubation with Zn or Cu decreased Cd uptake by the cells in a concentration-dependent manner. Cd accumulation data suggested that Cd is taken up by the cells via simple diffusion and carrier-mediated transport. Zn and Cu significantly increased the Km of Cd uptake with no or little effect on the Vmax. Pretreatment of the cells with ouabain and a metabolic inhibitor (FCCP) significantly decreased Cd accumulation, and coincubation with Zn or Cu led to an additional decrease in Cd accumulation by these cells. Overall, it seems that, in addition to simple diffusion, Cd is also taken up by LLC-PK1 cells via carrier-mediated mechanisms that are competitively inhibited by Zn and Cu.
Lead inhibited the production of nitric oxide (NO) in cytokine-induced macrophage cell lines of RAW264 and Mm1. The decrease was revealed in the concentration of lead (0.1-10 μg/ml) which did not decrease the viability of these cells. The inhibition was proved to be due to the suppression of an inducible type of NO synthase (iNOS) mRNA expression in RAW264 cells. This suppression was found at 24 h and then recovered to the control level (stimulated with cytokine alone) following culture. In the RAW264 cell line, the time course pattern of NO production corresponded to the iNOS mRNA expression. From these results, the inhibition of NO production by, at least in part, mRNA level could be implicated in lead-caused abnormalities in immune functions.
We found that AGL-517, an α-galactosylceramide (α-GalCer), possesses potent radioprotective activities against mice irradiated with 9 Gy of X-ray in contrast to its having no effect on mice irradiated with 10 Gy of X-ray. The result suggested the possibility that α-GalCers protect mice from bone marrow death. To examine this possibility, we examined the effects of two kinds of α- and β-GalCers on counts of palatelets (PLT) and white blood cells (WBC) in the peripheral blood of normal mice and mice irradiated in a whole body with 5 Gy of X-ray. α-GalCers significantly increased the PLT and WBC counts of both mice in comparison with the vehicle-treated group, and their potencies were stronger than those of their β-types. Furthermore, we evaluated the in vitro bone marrow cell-proliferation stimulatory activities of four kinds of GalCers, and found that α-GalCers show stronger stimulatory effects than β-types. These results demonstrate that the α-configuration of GalCers plays an important role in the manifestation of the above-mentioned activities of GalCers. The results also suggest that α-GalCers may be useful as hematopoietic stimulators as well as radioprotective agents.
O6-Benzylguanine (BG) is a potent depleter of a repair enzyme O6-alkylguanine-DNA alkyltransferase. Pretreatment of cells with BG potentiates the cytotoxicity of chloroethylating anti-center agents. In this study we used HeLa S3 cells to examine the cytotoxic potentiation of 39 compounds after BG pretreatment. Compounds tested included anti-cencer agents and carcinogens, and among them only the cytotoxicity of methylating, chloroethylating and fluoroethylating agents was potentiated. This in the first description of the cytotoxic potentiation of fluoroethylating agents. Potentiation ratios were found to vary even among compounds possessing the same alkylating group. By pretreatment with 10 μM of BG, the cytotoxicity of methylating agents such as N-methyl-N-nitrosourea and streptozotocine was potentiated 3.7 and 9.4 fold, respectively. For chloroethylating agents, the potentiation ratios were 3.5 for N-chloroethyl-N-nitrosourea, 8.6 for N-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-N'-(2-chloroethyl)-N'-nitrosourea (ACNU), 2.2 for N, N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), 3.0 for N-(2-chloroethyl)-N'-cyclohexyl-N-nitrosourea (CCNU) and 5.2 for chloroethyl methanesulfonate. With respect to fluoroethylating agents, the potentiation ratios were 7.2 for N-fluoroethyl-N-nitrosourea, 2.0 for N-cyclohexyl-N'-fluoroethyl-N'-nitrosourea and 5.5 for fluoroethyl methanesulfonate. No effect was observed with the bromoethylating agent, N-bromoethyl-N-nitrosourea. There was no potentiation of the cytotoxicity of anti-cancer agents such as mitomycin C (MCC), cisplatin (CDDP), 5-fluorouracil (5FU), bleomycin (BLM), prednisolone, camptothecin, etoposide, methotrexate or vinblastine. A possible mechanism for the cytotoxic potentiation of the test compounds by BG pretreatment is discussed.
To search for possible anti-tumor-promoters (cancer chemopreventive agents), we carried out primary screening of 23 triterpenoid hydrocarbons (1-23) isolated from ferns using an in vitro synergistic assay system. Of these triterpenoids, hop-17(21)-ene (2), neohop-13(18)-eme (3), neohop-12-ene (4), teraxerane (17), multiflor-9(11)-ene(18), multiflor-8-ene (19), glutin-5(10)-ene (21) and taraxastane (23) exhibited remarkable inhibitory effects on Epstein-Barr virus (EBV) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Further, compounds 2 and 3 exhibited remarkable anti-tumorpromoting effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test using 7, 12-dimethybenz[α]anthracene (DMBA) as an initiator and TPA as a promoter.
Brazilian propolis is known to induce the activation of murine effector cells. We isolated and identified six compounds from a water-soluble extract of Brazilian propolis, all of which have enhancing effects on the spreading and mobility of murine macrophages. These compounds were identified as caffeoylquinic acid-derivatives, namely, 5-caffeoylquinic acid (1), chlorogenic acid (2), 4-caffeoylquinic acid (3), 4, 5-dicaffeoylquinic acid (4), 3, 5-dicaffeoyquinic acid (5) and 3, 4-dicaffeoylquinic acid (6).
We studied the uptake mechanisms of anthracycline derivatives, pirarubicin (THP), daunorubicin (DNR) and doxorubicin (ADR), in K562 and multidrug-resistant K562/ADM cells, which overexpress a multidrug efflux pump P-glycoprotein (P-gp). The uptake of THP, DNR and ADR by K562 or K562/ADM cells was time-, tetmperature- and concentration-dependent. The THP and ADR uptake by the parental cells was not affected by treatment with 4 mM 2, 4-dinitrophenol (DNP) alone or DNP plus a P-gp specific inhibitor, cyclosporin A (CyA, 10 μM), while the DNR uptake in the DNP treatment group was significantly greater than that in the control group. These was no difference in the uptake of THP between DNP-pretreated K562 cells and DNP plus CyA-pretreated K562/ADM cells. The uptake of DNR or ADR was almost equal in both types of cell treated with DNP alone. Every kinetic constant for THP, DNR and ADR uptake by the sensitive cells was approximately equal to that in the resistant cells, respectively, under the above conditions. THP uptake was noncompetitively inhibited and stimulated on simultaneous treatment and preloading, respectively, of DNR or ADR in each type of cell. ADR showed noncompetitive inhibition of DNR uptake by either type of cell. Therefore, it was suggested that a common carrier-mediated transport system was involved in the uptake of THP, DNR and ADR, and that their binding sites in the carrier might be different from one another in both K562 and K562/ADM cells.
In order to introduce a suitable drug mixing base or covering for burns or decubital wounds, the usefulness of fiber extracted from the sweet potato was investigated. The healing effect of the fiber was evaluated by examining the extent of reduction in the size of wounds and changes in the quality of wounds in rats. In contrast to the control, the use of the fiber alone as a wound covering material reduced wounded areas by 21.0% at postoperative day 9, 19.5% at day 11, and 18.7% at day 13, and resulted in an obvious increase in the rats' weight. The number of days required for healing in all treated rats was 19 for the fiber groups and over 21 d for the control. In vitro, the fiber indicated excellent absorptive ability for serum and good adhesive ability for a number of proteins. This result suggested that the fiber has favorable properties for healing wounds which contain large amounts of exudate. Our macroscopic findings also indicated that the fiber protected wounds from dryness. These results suggested that sweet potato fiber could be use as a covering material in combination with drugs in skin wound therapy.
Environmental exposure to ultraviolet light B (UVB, wave lengths 290-320 nm) of the solar spectrum causes major damage, including an inflammatory response, in skin. In the present study, we estimated the ability of a stable derivative of ascorbic acid, ascorbic acid 2-O-α-glucoside (AA-2G), to reduce UVB damage, using the human keratinocyte cell line, SCC, established from squamous cell carcinoma. By pre- (9 h) and post-cultivation with AA-2G, a significant preventive effect on the decrease in the absolute number of surviving cells by exposure to UVB (typical dose, 20 mJ/cm2) was measurable by a neutral red-uptake assay. The release of leactate dehydrogenase from the cell membrane damaged by UVB was inhibited by AA-2G. In agarose gel electrophoresis, relatively high molecular weight DNA fragments were detected in irradiated cells after 6 h post-irradiation, suggesting that the mechanism of cell death was necrosis. Quantitative analysis of DNA content by flow cytometry indicated that AA-2G suppressed both an increase in debris with degraded nuclei and a decrease in cells in G1 and S phases, but not in the G2/M phase, by UVB exposure. These data suggest that AA-2G shows a photoprotective effect against UVB-induced damage in human epithelial cells.
The purpose of this study was to evaluate a simultaneous microdialysis method in blood and brain striatum to determine the relative pharmacokinetics and matabolism of L-3, 4-dihydroxyphenylalanine (L-dopa). L-Dopa (250 μmol/kg) was administered to rats with or without the aromatic amino acid decarboxylase (AADC) inhibitor carbidopa (25 μmol/kg) or benserazide (25 or 62.5 μmol/kg). L-Dopa, its metabolites, and AADC inhibitors in dialysates were analyzed by high performance liquid chromatography with an electrochemical detector. A moment analysis was also made to obtain pharmacokinetic parameters.After administration of L-dopa alone, it and its related metabolites were detected in both dialysates of blood and brain striatum. Coadministration of carbidopa (25 μmol/kg) or benserazide (62.5 μmol/kg) significantly enhanced the striatal amount of L-dopa by 8.0 and 6.1 times, respectively. Carbidopa and benserazide also increased striatal amounts of L-dopa metabolites, such as 3, 4-dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxy-4-hydroxyphenylethyleneglycol. Inhibition effect of benserazide on an extracerebral decarboxylation of L-dopa to dopamine (DA) was stronger than that of carbidopa. Carbidopa showed a higher striatal level of DA than benserazide. These results suggest a different effect of the two inhibitors on the DA formations in blood and brain striatum, and on the L-dopa transport through the blood-brain barrier (BBB).Thus, microdialysis is an easy and available method for simultaneously assessing the in vivo relative pharmacokinetics and metabolism of drugs in systemic circulation and a target organ.
We investigated the possible potentiation of the convulsive toxicity of enoxacin (ENX) by the concomitant topical application of a felbinac (FLB) patch in rats. A felbinac patch (Seltouch[○!R]; 0.5%, 3 cm×4 cm) was attached on the back of rats where their hair has been removed. ENX was infused from the left jugular vein at 8 h after the application of FLB patch under an unanesthetized and unstrained condition. Blood, CSF and brain samples were collected at the occurrence of convulsion, and ENX concentrations of each part were determined.As a result, no significant potentiation by FLB patch was found in the onset time of convulsion or in the ENX concentration of each part. Moreover, based on the assumption that there are no inter-species differences in ENX concentration in the brain at the occurrence of a convulsion (Cbr), the predicted plasma ENX concentration required to elicit convulsions in humans, which was estimated from the Cbr and Kp value of ENX in the brain of rats, was 20 times higher than the therapeutic plasma level.
The absorption of methochlorpromazine in rat small intestinal everted sac was investigated. 0.22% of the drug added in mucosal fluid was transferred to serosal fluid for 30 min at 37°C. The absorption of the drug was slightly inhibited by choline, and more markedly inhibited by more hydrophobic quaternary ammonium cations such as tetraethylammonium and cetyltrimethylammonium. Moderate inhibition was observed by a polyamine, spermine. The absorption rate of methochlorpromazine markedly depended on temperature. The Arrhenius plot of the apparent transfer rate constant revealed high activation energy (117 kJ/mol) for the transport. Uptake of methochlorpromazine to a small intestinal segment was also inhibited by other quaternary ammonium cations corresponding with their inhibitory effects on its transport. These results suggest that methochlorpromazine binds to the relatively hydrophobic region of small intestinal epithelial cells and transfers by passing through a high energy barrier.
N(α)-Phthalimidoglutarimide (thalidomide), 2-(2, 6-diisopropylphenyl)-1H-isoindole-1, 3-dione (PP-33) and its 4, 5, 6, 7-tetrafluoro derivative (FPP-33) augmented 12-O-tetradecanoylphorbol 13-acetate-induced production by human leukemia HL-60 cells of both tumor necrosis factor alpha (TNF-α) mRNA and secreted TNF-α protein. Intracellular TNF-α protein production was increased to a lesser extent.